Short elements with charged amino acids form clusters to sort protachykinin into large dense-core vesicles.
The sorting of neuropeptide tachykinins into large dense-core vesicles (LDCVs) is a key step in their regulated secretion from neurons. However, the sorting mechanism for protachykinin has not yet to be clearly resolved. In this study, we report that the clustered short elements with charged amino acids regulate the efficiency of protachykinin sorting into LDCVs. A truncation experiment showed that the propeptide and the mature peptide-containing sequence of protachykinin were sorted into LDCVs. These two regions exhibit a polarized distribution of charged amino acids. The LDCV localization of the propeptide was gradually decreased with an increasing number of neutral amino acids. Furthermore, the short element with four to five amino acids containing two charged residues was found to be a basic unit for LDCV sorting that enables regulated secretion. In the native propeptide sequence, these charged short elements were clustered to enhance the intermolecular aggregation by electrostatic interaction and produce a gradual and additive effect on LDCV sorting. The optimal conditions for intermolecular aggregation of protachykinin were at millimolar Ca(2+) concentrations and pH 5.5-6.0. These results demonstrate that the charged short elements are clustered such that they serve as aggregative signals and regulate the efficiency of protachykinin sorting into LDCVs. These findings reveal a novel mechanism for the sorting of neuropeptides into a regulated secretory pathway.
Ma
Guo-Qiang
GQ
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
Wang
Bin
B
Wang
Hai-Bo
HB
Wang
Qiong
Q
Bao
Lan
L
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D004583:Electrons; D006863:Hydrogen-Ion Concentration; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D015320:Tachykinins / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D010455:Peptides; D011498:Protein Precursors; D015320:Tachykinins; C054618:protachykinin; D002118:Calcium